The 3' ends of mRNAs are formed in eukaryotic cells either by the post-transcriptional processing of a primary RNA, or, less commonly, by the termination of transcription (Proudfoot et al, Transcription and Splicing, Eds Hames and Glover, IRL Press Limited (Oxford), pp. 97-99 (1988); Wickens, Trends Biochem. Sci., 15:277 (1990)). The 3' ends of the mRNAs of DNA viruses of most types are formed in a similar way, because most viruses employ the transcriptional apparatus of the cell to synthesize viral mRNAs.
The poxviruses differ from other DNA viruses in that they replicate in the cytoplasm of the cell, employing numerous viral enzymes, instead of host cell enzymes, to synthesize their RNAs. Poxviral proteins known to be involved in RNA synthesis and processing include a multisubunit RNA polymerase that resembles eukaryotic RNA polymerase II, several transcription factors, a capping-enzyme complex, an RNA methyltransferase, a poly(A) polymerase, and an endoribonuclease (Moss, Virology, Eds. Fields and Knipe, Raven Press (New York), pp. 2079-2112 (1990)). This assemblage of viral proteins suggests that the poxvirus may encode all the factors necessary for viral RNA synthesis, including those required for RNA 3' end formation.
The transcription of poxvirus genes is a temporally regulated process. Early genes are transcribed before viral DNA replication, intermediate genes are transcribed after the onset of viral DNA replication, and late genes are transcribed after the expression of the intermediate genes (Moss, Annual Rev. Biochem., 59:661 (1990)).
The processes used to form the 3' ends of viral RNAs are temporally regulated also. The 3' ends of the early RNAs are generated by the termination of transcription, which occurs about 50 nucleotides downstream of the signal sequence 5'UUUUUNU3' in the nascent RNA (Rohrmann et al, Cell, 46:1029 (1986); Shuman et al, J. Biol. Chem., 263:6220 (1988)). Interestingly, the process generating the termination of transcription of early genes does not appear to operate after the onset of viral DNA replication (Weir et al, J. Virol., 51:662 (1984); Weinrich et al, J. Virol., 61:639 (1987); Vos et al, EMBO J., 10:2553 (1991)). RNA transcripts of most characterized late genes appear to be heterogeneous in length, lacking the defined 3' ends characteristic of the early mRNAs (Mahr, et al, J. Virol., 49:510 (1984); Cooper et al, J. Virol., 37:284 (1981)). However, a few late transcription units whose RNAs are homogeneous in length have been identified. These include the cowpox virus gene encoding the most abundant viral protein, the major protein component of the A-type inclusion (ATI) bodies (hereinafter referred to as the ATI gene or 160K gene) (Patel et al, EMBO J., 6:3787 (1987); Patel et al, Virology, 149:174 (1986)); the equivalent vaccinia virus gene (hereinafter also referred to as the 94K gene) (Patel et al, Proc. Natl. Acad. Sci., 85: 9431 (1988); Amegadzie et al, Virology, 186:777 (1992)); and the telomeric transcription units of vaccinia virus, cowpox virus, and raccoon pox virus (Parsons et al, Virology, 175:69 (1990)).
Prior to the present invention, the mechanism involved in the generation of the defined 3' ends of late viral RNAs was not known. It has now been demonstrated that specific elements governing late transcription of poxvirus DNA can be used to direct RNA 3' end formation by site-specific RNA cleavage. In particular, it has now been demonstrated that the 3' ends of the late RNAs encoding the ATI protein are generated, not by the termination of transcription, as is the case for 3' end formation of early RNAs, but by site-specific cleavage of a precursor RNA transcript. This site-specific cleavage is effected by a poxvirus-induced factor, which factor forms part of the present invention.
The present invention makes possible site-specific RNA cleavage either in vitro or in vivo. In addition, the factor of the invention can be used in conjunction with other RNA processing enzymes to generate novel RNA molecules.